A site-specific labeling method was developed in which sulfhydryl groups of a murine IgG2a anti-ovarian monoclonal antibody, 5G6.4, were biotinylated with N-iodoacetyl-N′-biotinylhexylenediamine (Compound 1) following partial reduction of disulfide bonds with dithiothreitol. Reaction of 1-alkylated 5G6.4 with 125I-streptavidin gave immunoreactive streptavidin-1-biotinylated complexes. Radio-fast protein liquid chromatography data were consistent with the formation of a stable monovalent streptavidin-half-antibody complex as the major species. In vivo specific localization of these radioantibody conjugates to human tumor xenografts of ovarian carcinoma was confirmed by a comparative biodistribution study in nude mice using as a control the nonspecific 125I-streptavidin-1-alkylated UPC-10 (an irrelevant IgG2a monoclonal antibody) complex prepared analogously as described above. Tumor uptake for radiolabeled 5G6.4 [0.279 ± 0.041 % (SE) kg injection dose/g) was significantly greater [P < 0.025] than for UPC-10 [0.165 ± 0.027% kg injection dose/g]. The tumor:blood ratio (7.38 ± 1.285) for 5G6.4 was ≈3 times that for UPC-10 (2.48 ± 0.708, P < 0.01). This sulfhydryl site-directed approach demonstrated that reduced disulfides of monoclonal antibodies are viable sites for attaching labels without significant loss of in vitro and in vivo immunoreactivity.


Presented at the “Second Conference on Radioimmunodetection and Radioimmunotherapy of Cancer,” September 8–10, 1988, Princeton, NJ. Supported in part by NIH Grants ROI CA41531-02 and ROI CA42768-01A1.

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